1. Field of the Invention
The present invention relates to a magnetic sensor for target substance detection and its production method, and an apparatus and a kit for target substance detection using this sensor.
2. Description of the Related Art
As a quantitative immunoassay, a radiometric immunity analysis (RIA: radio immunoassay or IRMA: immunoradiometric assay) has been known for many years. This method labels a competitive antigen or an antibody by a radionuclide, and quantitatively measures the antigen from a measurement result of specific radioactivity. Thus, this method labels a target substance, such as an antigen, and measures this indirectly. Although this method has achieved large contribution in clinical diagnosis since sensitivity is high, there is a problem of safety of a radionuclide, and there is a defect that a facility and an apparatus for exclusive use are necessary. Then, methods of using labels, such as a fluorescent material, an enzyme, an electrochemical luminescence molecule, and a magnetic particle, have been proposed as methods of being easier to handle, for example. An enzyme immunoassay (EIA) which uses an enzyme as a label is a method of making an antigen-antibody reaction performed, making an enzyme-labeled antibody react, adding a substrate to the enzyme and making it color, and performing colorimetry with its absorbance.
Recently, a method of detecting easily a trace amount of magnetic particles used as a labeling substance is proposed by using a magnetoresistance effect film (refer to David R. Baselt et al., Biosensors & Bioelectronics 13, 731 (1998)) (hereafter, “Document 1”) and D. L. Graham et al., Biosensors & Bioelectronics 18, 483 (2003) (hereafter, “Document 2”).
In Document 1, two or more pieces of magnetic particles with 2.8 μm of diameter are detected using giant magnetoresistance effect (GMR) films in size of 80 μm×5 μm and 20 μm×5 μm. The magnetic film used in the GMR film is an inplane magnetization film, and a magnetic field applied to magnetic particles is applied in a direction perpendicular to a film surface of the magnetic film. Hence, as illustrated in FIG. 4, a stray magnetic field generated by the magnetic particles magnetized by an application of a magnetic field is applied to the magnetic film of the GMR film in an approximately in-plane direction, and magnetization of the magnetic film is aligned in this magnetic field direction. FIG. 4 illustrates a magnetic sensor 200, a magnetic particle 400, a magnetization vector 410, an applied magnetic field vector 420, and a stray magnetic field 430. Magnitude of electric resistance of a magnetoresistance effect film depends on a relative magnetizing direction of two magnetic films. That is, a parallel magnetizing direction makes electric resistance comparatively small and an anti-parallel magnetizing direction makes electric resistance comparatively large. In order to achieve magnetized states of the parallel and anti-parallel, the magnetoresistance effect film includes magnetic materials having such coercive forces that a magnetizing direction of a magnetic film in two magnetic films of the magnetoresistance effect film is fixed and a magnetizing direction of another film is magnetization reversible by a stray magnetic field from a magnetic particle. When a magnetic particle does not exist on a GMR sensor, since a magnetic field in an in-plane direction is not applied to a magnetic film even if an external magnetic field is applied, magnetization reversal does not occur. A detection circuit has construction that a bridge circuit includes fixed resistors, and two sensors, that is, a GMR sensor in which a magnetic particle is not immobilized, and a GMR sensor in which a magnetic particle can be immobilized, and a lock-in amplifier detects potential difference induced in this bridge circuit. In Document 2, a magnetic particle in 2 of diameter is detected using a GMR sensor in the size of 2 μm×6 μm. Similarly to Document 1, the GMR sensor is formed by arranging one in which a magnetic particle can be immobilized, and another in which a magnetic particle is not immobilized, and detects the magnetic particle by comparing output signals of these two GMR sensors. Nevertheless, a magnetic film is an inplane magnetization film, and a magnetic field applied to the magnetic particle is in an in-plane longitudinal direction to the magnetic film.
As described above, detection methods of a magnetic particle using a magnetoresistance effect film perform detection by magnetizing the magnetic particle in a desired direction, and changing a magnetizing direction of the magnetoresistance effect film by a stray magnetic field emitted from the magnetic particle. These methods have advantages that an apparatus, a reagent, and the like which are used for measurement are simply handled, and that it is detectable in a comparatively short time.
For example, when a target substance which is going to be detected is an antigen, immobilization of the magnetic particle on the sensor is performed using an antigen-antibody reaction. That is, a primary antibody is formed on the sensor, a specimen, such as blood which may contain an antigen, is made to react with the antibody, and then, the magnetic particle modified by a secondary antibody is made to react. Because of this series of operations, when the antigen exists in the specimen, bonding of primary antibody-antigen-secondary antibody-magnetic particle occurs on the sensor. If the antigen does not exist, the above-mentioned bonding cannot be performed and the magnetic particle is not immobilized on the sensor as a result.
Since one magnetic particle is immobilized on the sensor to one target substance when the above-mentioned immobilizing method is used, the one target substance is detectable by using the magnetic sensor with high sensitivity.
As labeling substances, various things, such as fluorescent substances, enzymes, electrochemical luminescent substances, radioactive substances, and magnetic substances, exist, and the number and concentration of target substances are detected using a detection unit suitable for a labeling substance.
When a fluorescence label, an enzyme label, an electrochemical luminescence label, or the like is used as a label in an optical measuring method, and detection of a target substance is performed by measuring an optical absorbance, a transmittance, or an amount of light emission. In addition, when using the radioactive label containing a radioactive isotope, specific radioactivity is measured and a quantitation of a target substance is performed. Using the above optical measurement methods and radiation measurement methods has a demerit that a large measuring apparatus is necessary. On the other hand, when a magnetic label proposed recently is used, detection using a small measuring apparatus is possible. In the case of using the magnetic label, a small magnetic sensor detects a magnetic field generated from a magnetic particle. As the magnetic sensor, a hall device or a magnetoresistive element is usable.
In the sensor in Document 2, a film with single composition is formed on the sensor, and substances with which target substances bond, for example, antibodies are arranged uniformly. Although size of a modified magnetic particle used for a biosensor is hundreds of nm to tens of μm, size of substances bonding with target substances, such as antigens, is several nm in many cases. Hence, as shown in FIG. 5A, if antibodies and the like are densely formed on the sensor, even if all the antibodies are absorbed in antigens, since a magnetic particle is far larger than an antibody, there arises a case that magnetic particles may not be immobilized to all the antigens. In consequence, there is a problem that, since the number of the immobilized magnetic particles is remarkably different from the number of the adsorbing antigens, there is a possibility that it becomes hard to detect the number or concentration of the antigens. For example, as illustrated in FIG. 5B, even when antigens are immobilized, the number of magnetic particles is not different from the case illustrated in FIG. 5A although the number of the antigens is different, and the number of magnetic particles and the number of antigens are not in one-to-one correspondence, and hence, the correct number of antigens is not detected.